Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues.
The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism / Q000494:pharmacology; D004136:Dinitrobenzenes; D005779:Immunodiffusion; D007202:Indicators and Reagents; D007700:Kinetics; D009578:Nitrobenzenes; D011485:Protein Binding; D011817:Rabbits; D012882:Skin Tests; D013050:Spectrometry, Fluorescence; D013434:Sulfenic Acids; D014364:Tryptophan
D004136:Dinitrobenzenes; D007202:Indicators and Reagents; D009578:Nitrobenzenes; D013434:Sulfenic Acids; C004788:2,4-dinitrobenzenesulfenyl chloride; C002732:2-nitrobenzenesulfenyl chloride; D014364:Tryptophan; D002772:Cholera Toxin; D000262:Adenylyl Cyclases